The invention relates to the field of microscopy, and particularly relates to the field of coherent anti-stokes Raman scattering microscopy.
Coherent anti-stokes Raman scattering (CARS) microscopy provides for the imaging of chemical and biological samples by using molecular vibrations as a contrast mechanism. In particular, CARS microscopy uses at least two laser fields, a pump electromagnetic field with a center frequency at xcfx89p and a Stokes electromagnetic field with a center frequency at xcfx89s. The pump and Stokes fields interact with a sample and generate a coherent anti-Stokes field having a frequency of xcfx89AS=2xcfx89pxe2x88x92xcfx89S in the phase matched direction. When the Raman shift of xcfx89pxe2x88x92xcfx89s is tuned to be resonant at a given vibrational mode, an enhanced CARS signal is observed at the anti-Stokes frequency xcfx89AS.
Unlike fluorescence microscopy, CARS microscopy does not require the use of fluorophores (which may undergo photobleaching), since the imaging relies on vibrational contrast of biological and chemical materials. Further, the coherent nature of CARS microscopy offers significantly higher sensitivity than spontaneous Raman microscopy. This permits the use of lower average excitation powers (which is tolerable for biological samples). The fact that xcfx89AS greater than xcfx89p, xcfx89S allows the signal to be detected in the presence of background fluorescence.
For example, U.S. Pat. No. 4,405,237 discloses a coherent anti-Stokes Raman spectroscopic imaging device in which two laser pulse trains of different wavelengths, temporally and spatially overlapped, are used to simultaneously illuminate a sample. The ""237 patent discloses a non-collinear geometry of the two laser beams and a detection of the signal beam in the phase matching direction with a two-dimensional detector.
U.S. Pat. No. 6,108,081 discloses a different method and apparatus for microscopic vibrational imaging using coherent anti-Stokes Raman scattering. In the apparatus of the ""081 patent, collinear pump and Stokes beams were focused by a high numerical aperture (NA) objective lens. The nonlinear dependence of the signal on the excitation intensity ensures a small probe volume of the foci, allowing three-dimensional sectioning across a thick sample. The signal beam is detected in the forward direction.
A prior art CARS imaging system (based on the ""081 patent) 10 is shown diagrammatically in FIG. 1, in which collinear pump and Stokes beams 12 at frequencies of xcfx89p and xcfx89S respectively, are directed to a microscope objective lens 16, and onto a sample 18. The CARS signal is detected in the forward direction, and is received by collecting optics 20, filtered by one or more filters 22, and detected by a detector 26.
The signal that is created in CARS imaging, however, includes a substantial amount of background with no vibrational contrast from which the signal must be filtered or somehow distinguished. For example, as shown in FIG. 2, a conventional lateral CARS intensity profile of a 535 nm polystyrene bead embedded in water includes a substantial amount of CARS background from water 30 in addition to the characteristic CARS signal from the bead 32. The presence of this background from the isotropic bulk water has hindered efforts to increase the sensitivity of CARS imaging, particularly in biological applications. The CARS background is caused by electronic contributions to the third order nonlinear susceptibility. There exists a non-resonant contribution to the CARS signal of the sample of interest as well as of the surrounding isotropic bulk medium (i.e., solvent), which is independent of the Raman shift, xcfx89pxe2x88x92xcfx89S.
For example, as shown in FIG. 3, a combined CARS image 40 and intensity profile 42 taken along line 44xe2x80x9444 of epithelial cells shows that the signal includes CARS background (as generally indicated at 46) that may not be easily distinguished from the microscopic sample signal (as generally indicated at 48). The lateral dimension (in xcexcm) is shown along the horizontal axis, and signal strength (in cts) is shown along the vertical axis. In certain embodiments, the non-resonant background of the sample and the solvent may overwhelm the resonant CARS signal of the sample.
One approach to reducing the non-resonant background field in CARS spectroscopy is to take advantage of the fact that the non-resonant background has different polarization properties than the resonant signal. In practice, this was done using non-collinear excitation beams with different polarization directions. For example, see Polarization-Sensitive Coherent Anti-Stokes Raman Spectroscopy, by Oudar, Smith and Shen, Applied Physics Letters, June 1979, pp.758-760 (1979); and Coherent ellipsometry of Raman Scattering of Light, by Akhmanov, Bunkin, Ivanov and Koroteev, JETP Letters, Vol.25, pp.416-420 (1977).
In high resolution CARS microscopy, however, tightly focused collinear excitation beams are necessary. It is known that tightly focusing polarized beams will result in polarization scrambling. See Principles of Optics, Born and Wolf, Pergaman Press, 1989, pp. 435-449.
There is a need, therefore, for a system and method for providing improved sensitivity of CARS microscopy, and in particular, to provide a CARS detection scheme that reduces the non-resonant background and hence yields a higher signal-to-background ratio.
The invention provides a system and method for detecting a nonlinear coherent field induced in a microscopic sample. The system includes in an embodiment, a first source for generating a first polarized electromagnetic field at a first frequency and a second source for generating a second polarized electromagnetic field at a second frequency that is different from the first frequency. The system further includes optics for combining the first polarized electromagnetic field and the second polarized electromagnetic field in a collinear fashion such that the difference in polarization angles is xcfx86 wherein xcfx86 is not equal to zero. The optics further direct the combined electromagnetic field toward a common focal volume. The system also includes a polarization sensitive detector for detecting a nonlinear coherent field that is generated responsive to the first and second polarized electromagnetic fields in the focal volume.